LCN2 induces DNA damage and increases ROS in normal, but not MF, CD34⁺ cells. (A) Treatment with LCN2 increased the proportion of normal, but not MF, CD34⁺ /γ-H2AX⁺ cells (repeated-measures ANOVA F test; **P < .01; n = 4). (B) Flow cytometric analysis of intensity of 2′,7′-dichlorofluorescein diacetate fluorescence, used to quantitate intracellular ROS, in normal and MF CD34⁺ cells (*P < .05, n = 5 and n = 6, respectively). (C) Flow cytometric analysis showing that treatment with MF CM increased the proportion of γ-H2AX⁺ normal BM MNCs to a similar degree as that induced by 20 ng/mL of LCN2. Prior incubation of an LCN2 antibody (R&D Systems) with MF MNC-CM reduced the proportion of γ-H2AX⁺ normal BM MNCs. (D) The increase in the proportion of normal γ-H2AX⁺ BM MNCs after the addition of LCN2 was not observed with the addition of an LCN2 antibody (repeated-measures ANOVA F test; P = .001; n = 4). (E-F) LCN2 treatment of normal BM CD34⁺ cells dramatically increased the percentage of CD34⁺annexinV⁺ cells (**P = .0081, ***P = .0008; n = 8); this increase was not observed with the addition of NAC to LCN2-containing cultures (repeated-measures ANOVA F test). (G-H) The percentage of MF annexinV⁺CD34⁺ cells was not increased after the addition of LCN2 or the addition of NAC to LCN2-containing cultures (repeated measures ANOVA F test; *P > .05; n = 6). NAC, N-acetylcysteine.